Cytotoxic drug conjugates

ABSTRACT

Conjugates of antibodies and cytotoxic methotrexate drugs make use of liners between the drug and antibody which are simple organic groups, wherein the link to the antibody is an acyl group and the link to the drug is an alkylidene hydrazide.

This application is a division of application Ser. No. 07/229,941, filedAug. 8, 1988 now U.S. Pat. No. 5,028,697.

FIELD OF THE INVENTION

The present invention belongs to the fields of organic chemistry,immunology, and pharmaceutical chemistry, and provides cytotoxic drugconjugates useful for the targeted administration of cytotoxic drugs topatients in need of such treatment. Targeting of the drug conjugates isobtained by the use of antibodies which recognize an antigen associatedwith the cell to be treated with the cytotoxic drug, and whichantibodies thereby carry the cytotoxic drug to the cell. Intermediatesused in the synthesis of the conjugates are also provided.

BACKGROUND OF THE INVENTION

As long ago as 1900, Ehrlich proposed that drugs might be guided to thetarget organ by attaching the drugs to substances which would seek thattarget organ. The technology to follow up on Ehrlich's suggestion didnot exist until monoclonal antibody technology began to appear in the1970's. For some years now, publications on the targeting of drugs withthe aid of antibodies have been regularly appearing. However as yet, noantibody-conjugated drug is approved for therapeutic use. Chemists andimmunologists continue to experiment, seeking a method of makingantibody-drug conjugates which will transport the drug reliably to thetarget organ and release it at the proper time. The present inventionprovides a series of methotrexate drug conjugates linked to antibodiesby means of a linker system which appears to provide particularly goodrelease of the drug.

SUMMARY OF THE INVENTION

The present invention provides a cytotoxic drug conjugate of the formula

    Ab[CO--X═N--HN--M].sub.m                               I

wherein m is an integer from 1 to about 10;

Ab is a physiologically-acceptable antibody or antigen-recognizingfragment thereof, which recognizes an antigen associated with anundesirable cell;

X is a linker of the formula ##STR1##

R is hydrogen, phenyl, or phenyl substituted with one or two nitro,halogen, cyano or trifluoromethyl groups;

Ar is ##STR2##

R¹ is hydrogen, amino, amino-C₁ -C₄ alkyl, hydroxy-C₁ -C₄ alkyl, orguanidino-C₁ -C₄ alkyl;

R² is (CH₂)_(n') ##STR3##

n is an integer from 0 to 5;

M is a methotrexate drug of the formula ##STR4##

R³ is ##STR5##

R⁴ is CO, SO₂, CO--(CH₂)_(s) or CO--NH;

R⁵ is hydrogen or C₁ -C₃ alkyl;

s is one or two;

Z is ##STR6##

t is an integer from 1 to 6;

u is an integer from 1 to 22;

R⁶ is hydroxy or a moiety which completes a physiologically-acceptablesalt.

The invention also provides a series of derivatized antibodies of theformula

    Ab[CO--X═R.sup.8 ].sub.m

wherein R⁸ is O, (OCH₃)₂ or (OCH₂ CH₃)₂, and X and m have theabove-stated meanings.

The invention also provides a method of controlling the growth of anundesirable cell which comprises administering a conjugate of theinvention parenterally to a patient, and pharmaceutical compositionscomprising a conjugate of the invention dispersed in aparenterally-administrable medium.

DETAILED DESCRIPTION OF THE INVENTION

The present drug conjugates are composed of an antibody, a linker and amethotrexate drug in the hydrazide form. The desirable therapeuticproperties of the conjugates are primarily obtained from the linker,which is so designed as to release the methotrexate drug from theantibody after the drug has been delivered to the vicinity of the targetcell by the antibody. The three major components of the conjugates willbe discussed individually, the synthesis of the conjugates will then beexplained, and, finally, examples of the synthesis and biologicaltesting of the conjugates will be shown.

The Antibody

The essential property of the antibody portion of the conjugates is itsability to recognize an antigen associated with an undesirable cell. Itwill be understood that the methotrexate drugs are highly cytotoxic to awide variety of cells, and that those drugs exhibit their full potencywhen internalized into the target cell. Thus, the antibody is preferablychosen for its ability to recognize, bind to and be internalized by thecell which is to be killed or otherwise controlled with the methotrexatedrug.

The source of the antibody is not critical to the present invention. Itmay be chosen from any class or subclass of immunoglobulin includingIgG, IgA, IgM, IgE and IgD. Similarly, the species of origin is notcritical so long as the antibody targets a cell where the effect of themethotrexate drug is useful.

In the present state of the art, monoclonal antibodies are most used indrug conjugates, and use of them is preferred in the present invention.However, polyclonal antibodies are not excluded. A newer type ofantibody is the chimeric antibody, which is prepared in the laboratoryby recombinant technology which permits expression of a modified DNAwhich encodes the antigen-binding region of any desired antibody, andalso encodes any other desired amino acid sequences. Thus, chimericantibodies of which one portion is derived from one species, and anotherportion is derived from another species may be obtained and used in thepresent invention.

The origin and nature of the antibody is not otherwise critical, so longas it targets the cell to be treated and is not, in itself, toxic to thepatient. Those of ordinary skill can readily prepare conjugates with acandidate antibody and evaluate them. Some discussion of the method ofevaluating antibodies and conjugates will be provided for convenience.First, the antibody should be produced by a hybridoma which issufficiently stable to allow preparation of reasonable quantities ofantibody. The antibody itself should be amenable to purification, and inparticular should be sufficiently water-soluble to allow chemicalmanipulations at reasonable concentration.

Conjugates prepared with the candidate antibody are first evaluated forantigen-binding capacity. A modest reduction from the binding capacityof the free antibody is expected and acceptable. Then, the conjugate istested to determine its in vitro cytotoxicity, against antigen positivecells. An effective conjugate can have cytotoxicity somewhat less thanthe free drug in the same assay. A conjugate which is accepted in thefirst two tests is then evaluated in a nude mouse human tumor xenograftmodel, as taught by Johnson and Laguzza, Cancer Res. 47, 3118-22 (1987).The candidate conjugate should be tested in nude mice against the freedrug, a mixture of free drug and free antibody, and a conjugate with anon-targeting immunoglobulin, and should exhibit improved potency orsafety over all. Dose ranging studies should be carried out in thexenograft model.

Conjugates which are potent in the xenograft model are submitted totests in animals which are known to express the antigen of interest in apattern similar to that seen in humans. If the conjugate produces asignificant degree of binding to the antigen in such tests, and if it isreasonably free of toxicity at doses predicted by the xenograft model tobe therapeutic, the candidate conjugate can be considered to havetherapeutic potential.

Many presently known antibodies are available for use in the presentinvention. The preferred specific antibody is L/1C2, which is producedby a hybridoma on deposit in the American Type Culture Collection,Rockville, Md., as HB9682.

Antibody 5E9C11, produced by an ATCC hybridoma, HB21, recognizestransferrin receptor, which is expressed by many tumors. An antibodycalled B72.3, available from the National Cancer Institute, recognizesantigens expressed by both breast and colon carcinoma.

Two interesting antibodies with reactivities against non-tumor antigensare OKT3 and OKT4, which bind to peripheral T-cells and human T-helpercells, respectively. They are produced by hybridomas on deposit in theATCC as CRL8001 and CRL8002, respectively.

Additional sources of antibodies useful for various therapeutic purposesare the following. Antihuman lymphocyte and monocyte antibodies, usefulfor immune modulation and tumor therapy, are produced by ATCC culturesHB2, HB22, HB44, HB78 and HB136. An antitransferrin receptor antibody,useful for tumor therapy, is produced by ATCC culture HB84. ATCC cultureHB8059 produces an antibody against colorectal carcinomamonosialoganglioside, and culture HB8136 produces an antibody againstmature human T-cell surface antigen, useful for immune modulation andT-cell leukemia therapy.

Further candidate antibodies are readily located by those of skill inthe art. A particularly useful source of information about readilyavailable antibodies is Linscott's Directory of Immunological andBiological Reagents, published by Linscott's Directory, 40 Glen Drive,Mill Valley, Calif. 94941. The 1984 edition lists more than 60tumor-associated monoclonal antibodies, and at least one commercialsource for each.

It will be understood that a variety of undesirable cells may be treatedwith conjugates of the present invention. The methotrexate drugs arewidely known to be active against the various types of cancer cells, andit is contemplated that cancer cells are among the preferred cells to betargeted by the present conjugates.

In particular, cells which support continued development of amalignancy, and cells of the immune system which control development ofanti-tumor immunity are also contemplated. Specific types ofcancer-related cells to be targeted include squamous carcinoma cells,adenocarcinoma cells, small cell carcinoma cells, glioma cells, melanomacells, renal cell carcinoma cells, transitional cell carcinoma cells,sarcoma cells, cells of supporting tumor vasculature, and cells oflymphoid tumors such as leukemias and lymphomas.

However, the methotrexate drugs are also cytotoxic to many other typesof cells. Thus, the conjugates can be used, by proper choice of theantibody, to kill or modify such undesirable cells as, for example,cells infected with virus particles, T cells infected with variousharmful agents, and cells of the immune system which promote or controldevelopment of autoimmune diseases.

It will be understood that properly chosen fragments of antibodies havethe same effect as the intact antibody. Thus, in the practice of thisinvention, antibody fragments, preferably F(ab')₂ fragments, whichrecognize an antigen associated with the cell to be treated, may be justas useful as are intact antibodies.

The exact mechanism by which the linker group reacts with and attachesto the antibody is not shown in formula I, and is not perfectly known.The reaction presumably is an acylation, as is demonstrated below, and anumber of locations on antibody molecules are subject to acylation. Mostcommonly, acylations of antibodies are thought to proceed on the freeamino groups of lysine moieties. However, the acylation can also attackhydroxy groups, phenol groups, imidazole rings and perhaps othermoieties.

Formula I indicates that from 1 to about 10 linker-drug moieties areattached to each molecule of antibody. Of course, the number of suchmoieties per antibody molecule is an average number because a givenbatch of conjugate will necessarily contain molecules having a range ofratios of drug-linker to antibody. The most efficient use of theexpensive antibody is obtained, of course, when a number of molecules ofdrug are attached to each antibody molecule. However, the attachment ofan excessive number of molecules of drug-linker moiety usually has anadverse effect on the antibody's ability to recognize and bind to itsantigen, so a compromise value for m must be found. In general, thepreferred value for m is from about 4 to about 10; another preferredvalue is from about 3 to about 8.

The Methotrexate Drug

The cytotoxic drug used in the present conjugates is methotrexate,aminopterin or a derivative thereof of formula VI, which compounds arecollectively called methotrexate drugs in this document. Themethotrexate drugs are used in the hydrazide form. The stereospecificforms of the various asymmetric centers of the drug are not indicated.Those of ordinary skill will understand that the stereochemistry of thedrug may affect its activity, as is clearly explained in the art. Theusual stereochemistry of the methotrexate drugs is preferably used inpreparing intermediates for the present conjugates, but formula VIincludes all stereochemical forms.

The bond between the methotrexate drug hydrazide and the linker is analkylidene hydrazide bond. It will be understood, however, that the bondmay exist in other forms in solution, particularly in physiologicalsolution. The double bond can be opened, allowing functional groups orprotons to bond to the nitrogen and carbon atoms. As a result, a hydroxygroup or other oxygen-linked species may attach to one side of theformer double bond, and amino-linked moieties may do so as well. Watercan weakly bond to one of the atoms. A moiety of the antibody also canform a weak bond to one of the atoms, and more than one of suchreactions may occur, forming mixtures. Such products are transitory,however, and throughout this document the conjugates will be describedas in the alkylidene hydrazide form, because that is the general andstable form of them.

In formula VI, the term C₁ -C₃ alkyl includes methyl, ethyl, propyl andisopropyl, preferably methyl or hydrogen.

The various groups which can vary in the drug of formula VI will bediscussed individually, and some preferred definitions of each will begiven. It will be understood that preferred methotrexate drug hydrazidesare made up of the preferred constituent groups, and pharmaceuticalchemists, having knowledge of the pertinent literature, can prepare anysuch drug hydrazide.

The group R³ is a bridging group which can be sulfur, oxygen, amino ormethylene, the latter two of which may be optionally substituted with C₁-C₃ alkyl. Typical such groups are amino, methylene, methylamino,propylamino, 1,1-propylene and 1,1-isobutylene. The preferred R³ groupsare amino and methylamino.

The group R⁴ is a bridging group which may be carbonyl, sulfonyl,acetyl, propionyl or carboxamido. In the latter three instances, thecarbonyl group is adjacent to the group Z. The most preferred R⁴ groupis carbonyl, and acetyl and propionyl are also preferred.

The group Z has an amino group at one end, which is attached to thegroup R⁴ and has a carbonyl or sulfonyl group at the other end, which isattached to the ═N--HN group. The Z group of formula VII is derived fromglutamic acid and, when t is not 1, constitutes the residue ofpolyglutamic acid. The preferred Z group of formula VII, however, isthat wherein t is 1.

The Z group of formula VIII terminates in a sulfonyl, and accordingly isthe residue of the corresponding sulfonic acid. The group of formulaVIII has one or two methylene groups, preferably two.

The Z group of formula IX is an amino acid of variable length, which maycontain from 1 to 22 methylene groups. A preferred class of groups offormula IX contains from 1 to 10 methylene groups, more preferably from3 to 8 methylene groups.

The most preferred Z groups are those of formula VII, wherein t is 1,and those of formula IX, wherein u is from 3 to 8.

In the groups of formula VII, VIII and IX, a carboxy group R⁶ ispresent, in either free or salt form. The salts are formed with anymoiety capable of forming a physiologically-acceptable salt of thecarboxylic acid. Alkali metal and hydrohalide salts are particularlyappropriate. Thus, the sodium, potassium and lithium salts, as well asthe hydrochloride, hydrobromide and hydrofluoride salts, areparticularly useful in the practice of the present invention. Othersalts acceptable in pharmaceutical chemistry, however, are also useful.For example, amine salts such as triethylamine, triethanolamine,ethyldimethylamine and the like are useful, as are quaternary ammoniumsalts including tetraalkylammonium salts, (benzyl orphenyl)trialkylammonium salts and the like. Among ammonium salts,tetrabutylammonium, benzyltrimethylammonium, and tetramethylammonium aretypical and preferred salts. Pharmaceutical chemists continually usesalts of carboxylic acids, however, and the present salts, wherein R⁶ isa salt-forming moiety, may be prepared with any base which forms aphysiologically-acceptable salt.

The necessary intermediates from which to derive groups of formula VIare in the pharmaceutical chemical art, and those of ordinary skill inthat art can obtain any of them. A useful review article on thesynthesis of methotrexate drugs is Rahman and Chhabra, The Chemistry ofMethotrexate and its Analogues, Medicinal Res. Rev. 8, No. 1, 95-155(1988).

The Linker

The linker group X is an organic group which is bonded to the carbonylat one end, and to the hydrazide at the other.

The linker group of formula II is an alkylene group, wherein the moiety(CH₂)_(n) is, for example, methylene, ethylene, propylene, pentylene andthe like.

The group R is hydrogen or phenyl, which may be substituted with one ortwo electron-withdrawing groups. Thus, the group R may be phenyl,3-nitrophenyl, 2,4-dichlorophenyl, 3-bromo-5-fluorophenyl,4-cyanophenyl, 3-trifluoromethylphenyl,2-chloro-4-trifluoromethylphenyl, 3-nitro-4-trifluoromethylphenyl andthe like. The term halogen is used in this document to refer to chloro,bromo and fluoro. Hydrogen is a preferred R group; unsubstituted phenylis another preferred R group, and a third preferred R group ismono-substituted phenyl.

The group of formula III is an alkylaryl moiety wherein the bridgingaryl group Ar is phenyl or pyrrolyl. A phenyl Ar group is linked at themeta or para position, preferably at the para position. The moiety(CH₂)_(n) in the group of formula III is the same as the correspondinggroup in formula II; thus, when n is 0, the group is nothing more than abond, or it may be an alkylene group as described above. It is preferredfor n to be 0; it is also preferred for n to be 2-4.

The group of formula IV is an amide-bonded linker, which may contain oneor two alkylene bridges. The alkylene groups, (CH₂)_(n) in the group offormula IV are the same as those discussed above under the group offormula II. In the design of groups of formula IV, the two optionalalkylene bridges may be considered independently--that is, n may be 0 inboth instances, one may be 0 and the other may be an integer from 1 to5, or both may be integers from 1 to 5. It is preferred that both of thegroups (CH₂)_(n) be alkylene groups wherein n is from 1 to 4, morepreferably 2 or 3.

The group of formula V is a more complex amide-linked group, oftenincorporating an amino acid moiety. The group (CH₂)_(n) is similar tothe same group as discussed under formula II. The pendant group may be aC₁ -C₄ alkyl group, substituted with amino, hydroxy or guanidino(NH--C(═NH)--NH₂). Thus, it includes groups such as aminomethyl,2-aminoethyl, 4-aminobutyl, 2-hydroxyethyl, 3-hydroxypropyl,4-hydroxybutyl, guanidinomethyl, 2-guanidinoethyl, and 4-guanidinobutyl,for example. R¹ may also be hydrogen or amino. Preferred R¹ groups areamino, hydroxymethyl, aminoalkyl, especially 4-aminobutyl, andguanidinoalkyl, especially 4-guanidinobutyl.

The group R² is a bond when n is 0, or may be an alkylene group of 1 to5 carbon atoms, phenyl or pyrrolyl. A phenyl R³ group is linked at themeta or para position. A (CH₂)_(n) group is the same as thecorresponding alkylene group described under the group of formula II,and may have the same values.

The most preferred linker groups of formulae II, III, IV and V are4-benzylidene, 4-methylbenzylidene, and2-ethylaminocarbonyl-4-benzylidene.

The Derivatized Antibodies

The derivatized antibodies are intermediates for the synthesis of theconjugates, consisting of the antibody modified by reaction with thelinker intermediate. The linker group is terminated with a carbonyl or adi(methyl or ethyl) acetal thereof, with which the methotrexate drughydrazide is reacted in a final step. Thus, the X group of the linkerterminates with ═O or (OCH₃)₂ or (OCH₂ CH₃)₂ in the derivatizedantibodies. The carbonyl form is preferred but the acetals aresatisfactory reactants and may be used when it is convenient to do so.

The variables X and m have the same values in the derivatized antibodieswhich have been discussed above. The preferred derivatized antibodiescorrespond to the preferred conjugates, in that they are comprised ofthe preferred antibodies, combined with the preferred number m of linkergroups made up of the preferred X groups.

Synthesis

The conjugates of the present invention are prepared according toprocesses which are, in general, similar to those presently used in theart. It is preferred to react the linker with the antibody first, andthen to react the linker with the methotrexate drug hydrazide as thefinal step. An advantage of that synthetic procedure is that thestereospecific form of the methotrexate drug is most readily preserved.

Thus, the first step or steps of the synthesis is to prepare the linker,in an appropriate form to be reacted with the antibody at one end, andwith the methotrexate drug at the other. Then, the linker is attached tothe antibody to prepare the derivatized antibody, and, finally, themethotrexate drug is attached to the carbonyl or acetal end of thelinker. It will be understood that, whenever the antibody is present ina reaction mixture, the process must be carried out under appropriatelymild conditions to prevent damage to the antibody.

The synthesis may be represented schematically as follows.

    Ab+m[R.sup.7 --CO--X═R.sup.8 ]→Ab[CO--X═R.sup.8 ].sub.m

    Ab[CO--X═R.sup.8 ].sub.m +m[H.sub.2 NHN--M]→Ab[CO--X═N--HN--M].sub.m.

wherein R⁷ is a carboxylic acid activating group;

and Ab, X, R⁸ and m are as defined above.

In the above formula, the group R⁷ is chosen from among well-knowngroups customarily used to activate carboxylic acids for use asacylation reagents. For example, succinimidoxy, phthalimidoxy,methanesulfonyloxy, toluenesulfonyloxy, benzenesulfonyloxy,benzotriazolyloxy, chloro, bromo, azido and the like are commonly usedas such activating groups. The preferred activating groups aresuccinimidoxy, phthalimidoxy and benzotriazolyloxy.

While the reactions are carried out, free amino groups which are part ofthe pendant R¹ substituent are blocked with conventional, readilyremoved protecting groups. Such groups are exhaustively discussed byGreen, Protective Groups in Organic Synthesis, John Wiley & Sons, N.Y.,1981. The groups used here must be removable under quite mild conditionsto avoid damage to the antibody. Such protecting groups are known tochemists; particularly useful ones include ethyl acetoacetate,citraconate, and ethyl dimethylmaleate.

The linker intermediates, R⁷ --CO--X═R⁸, are comparatively simplecompounds and can be purchased, or readily prepared by conventionalmethods. Those linker intermediates having a carboxamido linkage aremost conveniently prepared by reacting a primary amine, forming one endof the linker intermediate, with an activated carboxylic acid, formingthe other end of the intermediate. Such reactions can be carried out inmild aqueous base at ambient or slightly elevated temperature, andproceed in good yields in a few hours time.

The various steps of the synthesis of the present conjugates can beoperated to maximize throughput of the equipment in which the process iscarried out, or to maximize yield. In most if not all cases, theantibody itself will represent the largest cost in the process, andtherefore optimization of the process will call for maximizing yieldbased on the antibody. The exact optimum operating conditions,therefore, will depend on the conditions of maximum stability of theparticular antibody in use. Accordingly, it is probable that optimumoperating conditions will demand the use of quite a large excess ofmethotrexate drug in order to utilize the antibody to the maximum whileminimizing the length of time in which the antibody is exposed to thereaction mixture.

The carboxylic acid activating groups are readily placed on thecarboxylic acids of the linker intermediates by use of, for example,dicyclohexylcarbodiimide or other esterification reagents. Suchreactions are carried out in inert organic solvents, such as dioxane,tetrahydrofuran, chlorinated hydrocarbons and the like, and may beperformed at moderate temperatures in the range of about 0°-50° C.Preparation of the linker intermediates is further explained below inthe Preparations.

The primary concern in choosing the conditions under which to react thelinker intermediates with the antibody is maintaining the stability ofthe antibody. The reaction must be carried out in aqueous medium of acomposition which will not harm the antibody. A particularly suitableaqueous medium is a borate buffer solution, in which the concentrationof borate ion is in the range of about 0.1-0.5 molar. Anotherappropriate aqueous medium in which to carry out the reaction isphysiological buffered saline solution. The pH of the reaction mediumshould be slightly basic, in the range of about 7-9. While the reactionmedium should be aqueous, the presence of small amounts of organicsolvents is not harmful, and may be quite convenient. For example, itmay be most advantageous to dissolve the linker intermediate in a smallamount of organic solvent, for example, dimethylformamide, acetonitrile,tetrahydrofuran, dioxane, or a glycol ether, and add the organicsolution to the antibody solution in the aqueous medium.

In general, it will be necessary to operate the reaction at acomparatively low concentration because the solubility of antibodies isgenerally not great. For example, the concentration of the antibody isusually in the range of about 5-25 mg per ml of aqueous medium.

As described above, from 1 to about 10 moles of linker and drug areattached to each mole of antibody. In order to obtain that conjugationratio, it is usually necessary to use an excess quantity of linkerintermediate. The reactivity of antibodies and active esters underacylating conditions is somewhat variable, but in general, from about 5to about 15 moles of linker intermediate per mole of antibody are usedin the process.

The acylation reaction is allowed to proceed from a few minutes to a fewhours, at temperatures in the range from about 0° C. to about 40° C.Obviously, elevated temperatures may be injurious to the antibody and itis more advisable to operate at low temperatures, particularly since thereaction is inherently quick.

When the derivatized antibody, having the linker intermediate groups inplace, has been prepared, the reaction mixture can be chromatographed byconventional procedures, as shown in the examples below, to separate thederivatized antibody from unreacted linker intermediate. If thepurification is not done at this point then methotrexate drug will bewasted by reacting it with the excess linker intermediate in the firstreaction mixture.

Finally, the drug conjugate is completed by reacting the γ-hydrazide ofthe methotrexate drug with the derivatized antibody. The reaction is asimple one of the ketone or aldehyde, or acetal thereof, which is theterminating group of the linker intermediate, with the drug hydrazide.It is a quick reaction which goes well at approximately ambienttemperature in aqueous media. Ideally, the acylated antibody is purifiedby chromatography, eluting with a solution which forms a satisfactoryreaction medium for the methotrexate drug reaction, as well as providinggood separation in the chromatography. Dilute aqueous buffers areappropriate for both purposes.

For example, a particularly useful reaction medium is dilute acetatebuffer, more particularly, 0.1 molar sodium acetate at a slightly acidpH in the range of from about 5 to about 7. The reaction also may becarried out, however, in borate buffer, slightly acid phosphate buffers,physiological buffered saline and the like, so long as the pH isslightly acid. Small amounts of organic solvents in the reaction mediumare not harmful, as discussed above, so long as the solvents do not havea tendency to damage the antibody.

The reaction with the methotrexate hydrazide is carried out for reactionperiods in the range from about an hour to about a day. Reactiontemperatures in the range of about 0°-40° C. are used, choosing thetemperature to maximize the yield based on the amount of antibody in thereaction mixture.

Finally, the drug conjugate of the present invention is purified andisolated by chromatography by conventional means. It is particularlyconvenient to elute the drug conjugate with physiological bufferedsaline. It may be possible to elute the drug conjugate at aconcentration which is appropriate for administration to the patient,but it will usually be necessary to concentrate the conjugate, as byvacuum dialysis.

The synthesis of the drug conjugates of the present invention is furtherexplained by the following specific Preparations and Examples.

Preparation 1

Methotrexate-γ-hydrazide

To a 100 ml flask was added 1.61 g (10 mmoles) of L-glutamic acid,5-methyl ester. To it was added 60 ml of t-butyl acetate and the mixturewas stirred briefly. To it was then added, dropwise, 1.58 g (11 mmoles)of 70% perchloric acid, with good stirring. The mixture was allowed tostir under nitrogen for two days, and it was then extracted with 100 mlof 0.5N hydrochloric acid in three portions. The aqueous layers werecombined and neutralized with 30 g of sodium bicarbonate. The neutralsolution was extracted with three portions of diethyl ether, 150 ml intotal, and the organic layers were combined and washed with brine. Thewashed organic solution was then dried with sodium sulfate andevaporated at ambient temperature under vacuum to obtain 1.18 g (5.4mmoles) of a clear oil which was identified as L-glutamic acid,5-methyl-1-t-butyl diester.

To an oven-dried 100 ml flask were added 0.92 ml (6.6 mmoles) oftriethylamine, 1 ml (6.6 mmoles) of diethyl cyanophosphonate and 49 mlof dimethylformamide, freshly distilled under vacuum from barium oxide.To the stirred solution was added 506 mg (1.3 mmoles) of2,4-diamino-6-[N-methyl-N-(4-carboxyphenyl)amino]pteridine trihydrate.When the intermediate had dissolved with stirring, the mixture washeated to 80° C., and then 0.20 ml (1.4 mmoles) of triethylamine and 342mg of the L-glutamic acid diester prepared above were added in 1 ml ofdimethylformamide. The mixture was stirred for two hours at 80° C., andwas then cooled and the solvent was removed under vacuum. The residuewas taken up in 300 ml of chloroform, and was washed with 5% sodiumbicarbonate solution. The aqueous layer was extracted with chloroform,and the organic layers were combined, dried over sodium sulfate, andconcentrated under vacuum to obtain 1.35 g of orange oil, which waschromatographed on 150 g of silica gel, eluting with 10% methanol inchloroform. The product-containing fractions were combined andconcentrated to obtain 645 mg of methotrexate diester, where theγ-carboxy was in the methyl ester form and the α-carboxy was in thet-butyl ester form.

The above intermediate was combined with another lot of the same, making697 mg (1.3 mmoles) in all, and was dissolved in 12 ml of methanol. Toit was added 170 mg (5.3 mmoles) of anhydrous hydrazine, and the mixturewas stirred at ambient temperature under nitrogen for six days. Then thesolvent was removed under vacuum, and the residue was chromatographed on150 g of silica gel, eluting with 15% methanol in chloroform, to obtain470 mg (0.9 mmoles) of methotrexate-α-t-butyl ester-γ-hydrazide.

The above intermediate was dissolved in 120 ml of 1N hydrochloric acid,and was heated at 55° C. for 50 minutes. It was then concentrated undervacuum to a solid, and the residue was taken up in 80 ml of 0.01Mammonium acetate at pH 8. The solution was stored for three days at 4°C., and was then chromatographed on 300 ml of Sepharose Fast Flow Q,(Pharmacia, Inc., Piscataway, N.J.) eluting in a gradient manner withthe same buffer used immediately above (Buffer A) and 1.0M ammoniumacetate at pH 8 (Buffer B). The product-containing fractions eluting in100% Buffer B were combined and repeatedly lyophilized to remove tracesof ammonium acetate. The yield was 326 mg (0.7 mmoles) ofmethotrexate-γ-hydrazide.

Preparation 2

Production of L/1C2 antibodies

Vials of frozen L/1C2 hybridomas are obtained from the American TypeCulture Collection, under the accession number HB9682. Viable cells arerecovered by thawing the contents of a vial in a 37° C. water bath whileswirling the vial. The cell suspension is then diluted 1:2 with balancedsalt solution (Grand Island Biological Company (GIBCO), 3175 StaleyRoad, Grand Island, N.Y. 14072) and the suspension is centrifugedthrough a serum underlay to partition the cells from the cryogenicmedium. The supernatant is aspirated, and the cells in the cell pelletare suspended in culture medium (Ventrex HL-1, Ventrex Laboratories,Portland, Me.) supplemented with 10% fetal calf serum, 2 mM L-glutamine(GIBCO) and 50 ug/ml gentamicin sulfate (GIBCO)) in T75 tissue cultureflasks, in 5% carbon dioxide at 37° C. Supernatants from nearlyconfluent cultures are collected and residual cells are removed bycentrifugation. Antibody is purified from the cell free supernatant bypassing over a Protein A Sepharose column (Pharmacia). Antibody binds tothe column and culture medium is washed free in 0.01M sodium phosphateat pH 8.0. Antibody is then eluted from the column with 0.1M sodiumphosphate buffer at pH 3.5. Eluted antibody is immediately neutralizedwith 1M Trizma buffer (Sigma, St. Louis, Mo.) at pH 7.4 and dialyzed andconcentrated in a vacuum dialyzer (Bio-Molecular Dynamics, Beaverton,Oreg.) containing 0.01M sodium phosphate pH 7.4 plus 0.15M sodiumchloride. Antibody preparations are sterilized by filtration through 0.2μm pores and stored at 4° C. until used.

Preparation 3

3-(4-Formylphenylcarbonylamino)propionic acid, N-succinimido ester

To a 250 ml flask were added 3 g (20 mmoles) of 4-carboxybenzaldehydeand 2.3 g (20 mmoles) of N-hydroxysuccinimide in 100 ml of dioxane. Themixture was stirred for 5-10 minutes, and then 4.1 g (20 mmoles) ofdicyclohexylcarbodiimide was added. The mixture was stirred for one hourat ambient temperature, and was then filtered. The filtrate wasevaporated under vacuum to obtain 9.4 g of a white solid, which wasrecrystallized from 25 ml of hot isopropanol. The intermediate productwas triturated with isopropanol to obtain 2.1 g (8.5 mmoles) of thedesired N-succinimido ester of 4-carboxybenzaldehyde.

Additional batches of intermediate were made, and 10 g (40 mmoles) totalof the N-succinimido ester was added to a solution of 3.6 g (40 mmoles)of β-alanine in 40 ml of 1N sodium hydroxide and about 100 ml of water.The pH was kept above 8 while the mixture was stirred for 1.5 hours. Themixture was then filtered, and the filtrate was made acid to pH 1.9 with2N hydrochloric acid. It was extracted three times with 150 ml total ofethyl acetate, and the organic layers were combined and washed withbrine. The organic layer was then dried over sodium sulfate andevaporated under vacuum to obtain 4.6 g (21 mmoles) of a white solid,3-(4-formylphenylcarbonylamino)propionic acid.

One hundred mg (0.45 mmoles) of the above intermediate, 103 mg (0.5mmoles) of dicyclohexylcarbodiimide, 57.5 mg (0.5 mmoles) ofN-hydroxysuccinimide and 10 ml of dioxane were added to a small flask,and the mixture was stirred at ambient temperature under nitrogen. Theprogress of the reaction was observed by thin layer chromatography, and75 mg (0.36 mmoles) of additional dicyclohexylcarbodiimide and 45 mg(0.4 mmoles) of additional N-hydroxysuccinimide were added. After fourhours, the reaction mixture was filtered, and the filtrate wasevaporated to a solid under vacuum. About 200 mg of impure product wasobtained, which was chromatographed on 30 g of silica gel, eluting with5% isopropanol in dichloromethane. The product-containing fractions werecombined and evaporated to obtain 81 mg (0.25 mmoles) of the desiredintermediate in somewhat impure form.

Preparation 4

Antibody L/1C2 propionyl-3-aminocarbonyl-4-benzaldehyde

To a 100 ml flask at ambient temperature was added a solution of 1026 mg(6.8 μmoles) of antibody L/1C2 in 76.1 ml of 0.34M borate buffer at pH8.6, followed by 14.1 mg (44 μmoles) of the active ester fromPreparation 3, in 3.3 ml of acetonitrile. The mixture was stirred forone hour at ambient temperature. It was then chromatographed on 90 g ofSephadex G25 (Pharmacia), eluting with 0.1M sodium acetate at pH 5.6.The fractions were evaluated by ultraviolet analysis at 258 and 280 nm,and the product-containing fractions were combined to obtain 948 mg (6.3μmoles) of the desired product, in the form of 111.5 ml of solutionhaving a concentration of 8.5 mg/ml. The conjugation ratio of thederivatized antibody was 4.8 moles of linker per mole of antibody.

EXAMPLE 1 Conjugate of antibody L/1C2 propionyl-3-aminocarbonyl-4-benzaldehyde with methotrexate-γ-hydrazide

A 55.6 ml portion of the product of Preparation 4, containing 472 mg(3.1 μmoles) of the derivatized antibody, was diluted with 87 ml ofadditional 0.1M sodium acetate at pH 5.6.

A 101 mg (216 μmoles) portion of methotrexate-γ-hydrazide was taken upin 7.2 ml of acetonitrile and 21.6 ml of 1M monobasic potassiumphosphate buffer. A little 5N sodium hydroxide was added to thesolution, and the solution was then added to the antibody solution. Thereaction mixture was made acid to pH 5.8 with glacial acetic acid, andwas stirred for 16 hours at ambient temperature. It was thencentrifuged, and the supernatant chromatographed on two 90 g SephadexG25 chromatography columns, eluting with physiological buffered saline.

A total of 202.8 ml of solution was collected from the chromatography,which was analyzed by ultraviolet spectroscopy, observing the spectrumat 280 and 370 nm. Analysis showed that the solution contained 0.018mg/ml of methotrexate, and 1.87 mg/ml of antibody. The conjugationratio, thus, was 3.0 in molar terms.

The product solution was concentrated by vacuum dialysis againstphysiological buffered saline in the cold, combining the productsolution of this example with 212 ml of product solution from a similarrun. The volume was reduced by dialysis to 108 ml.

The product was evaluated against the human tumor T222 (Masui et al.,Cancer 44, 1002-07 (1984)), established in female nude mice asxenografts. The conjugate was administered to the mice at the doses(based on content of methotrexate hydrazide) shown in the table below,as intravenous injections, on days 3, 6 and 9 after the tumors wereimplanted in the mice. The tumors were measured two, three and fourweeks after implantation of the tumors, and the results are reportedbelow as the percent inhibition of tumor growth in the treated mice,compared to the growth in untreated control animals. Positive controlanimals were treated with methotrexate-γ-hydrazide in uncombined form,to provide a comparison with the conjugate-treated animals. Toxicity ofthe treatments is reported as the number of animals in each 5-mousetreatment group, which exhibited signs of toxicity. The results were asfollows.

                                      TABLE 1                                     __________________________________________________________________________                   14 Days   21 Days   28 Days                                    Treatment                                                                              Dose  Inhibition                                                                         Toxicity                                                                           Inhibition                                                                         Toxicity                                                                           Inhibition                                                                         Toxicity                              __________________________________________________________________________    Example 1                                                                              4 mg/kg                                                                              91% 3     100%                                                                              3     100%                                                                              3                                              2     86   0    98   0    100  0                                              1     94   0    97   0    98   0                                              0.5   11   0    -27  0     0   0                                     MTX Hydrazide                                                                          20    40   0    25   0    14   0                                              4     17   0     5   0    13   0                                              1     11   0     9   0    32   0                                     __________________________________________________________________________

EXAMPLE 2 Conjugate of antibody L/1C2propionyl-3-aminocarbonyl-4-benzaldehyde with methotrexate-γ-hydrazide

The process of Example 1 was repeated four times, under differentconditions as follows.

A. A 0.67 mg portion of methotrexate-γ-hydrazide, dissolved in 67 μl of0.1M sodium acetate at pH 5.6 and 33 μl of acetonitrile, was combinedwith 1 ml of solution containing 2.2 mg of the intermediate ofPreparation 4 in 0.1M sodium acetate, and the mixture was allowed tostand for 11 hours. It was then chromatographed on 5.9 g of Biogel P6(Bio-Rad Laboratories, Richmond, Calif. 94804) to obtain 5 ml ofconjugate solution, which was found to contain 0.6 mg of the desiredconjugate, at a conjugation ratio of 3.2 moles of methotrexate per moleof antibody.

B. The same amount of methotrexate-γ-hydrazide solution used in A wascombined with 0.95 ml of solution of the intermediate of Preparation 4,containing 0.42 mg of the intermediate and the mixture was allowed tostand for 11 hours. It was chromatographed as in A to obtain 4.75 ml ofconjugate solution, containing 0.11 mg of conjugate at a conjugationratio of 5.4 moles per mole.

C. An 0.7 mg portion of methotrexate-γ-hydrazide dissolved in 50 μl ofacetonitrile and 100 μl of 1M phosphate buffer at pH 5.8 was combinedwith 2.2 mg of the intermediate of Preparation 4 dissolved in 1 ml of0.1M sodium acetate at pH 5.6. When the mixture had stood for 11 hours,it was chromatographed as in A to obtain 5.3 ml of conjugate solutioncontaining 1.3 mg of conjugate at a conjugation ratio of 3.3 moles permole.

D. An 0.7 mg portion of methotrexate-γ-hydrazide, dissolved in 50 μl ofacetonitrile and 150 μl of 1M phosphate buffer at pH 5.8 was combinedwith 2.2 mg of the intermediate of Example 4, in 1 ml of 0.1M sodiumacetate. The mixture was allowed to stand for 14 hours, and was thenchromatographed as in A to obtain 5.3 ml of conjugate solution,containing 1.7 mg of conjugate at a conjugation ratio of 3.3.

The products of processes C and D above were evaluated for their abilityto inhibit the growth of cells of tumor T222 in tissue culture, byadding controlled concentrations of the conjugates to the culturemedium. Free antibody L/1C2, used as a control, inhibited growth of thecells by 37% at 16 mcg/ml, and by 98% at 160 mcg/ml. The conjugate ofExample C above inhibited growth by 13% at 0.0013 mcg/ml, and by 87% at0.013 mcg/ml, based on the content of methotrexate-γ-hydrazide.

The conjugate of Example D above inhibited growth of the cells by 21% at0.0016 mcg/ml, and by 92% at 0.016 mcg/ml, based onmethotrexate-γ-hydrazide content.

Preparation 5

Antibody L/1C2 carbonyl-4-benzaldehyde

A 0.31 mg portion of 4-carboxybenzaldehyde, N-succinimido ester, wasprepared as described in Preparation 3 above. It was dissolved in 100 μlof dimethylformamide and was added to 18.9 mg of antibody L/1C2 in 2.1ml of 0.34M borate buffer at pH 8.6. The mixture was stirred for 1.5hours at ambient temperature, and it was then chromatographed on 6 g ofBiogel P6, eluting with 0.1M sodium acetate at pH 5.6. A 6.2 ml portionof solution was obtained, and was analyzed by ultraviolet, observing thespectrum at 256 and 280 nm. The analysis indicated that 16.1 mg ofderivatized antibody was obtained, at a concentration of 2.6 mg/ml,having a conjugation ratio of 5.2 moles of linker per mole of antibody.

EXAMPLE 3

Conjugate of antibody L/1C2 carbonyl-4-benzaldehyde withmethotrexate-γ-hydrazide

Four ml of the solution from Preparation 5, containing 10.4 mg (0.069μmoles) of derivatized antibody, was combined with 3.2 mg (6.8 μmoles)of methotrexate-γ-hydrazide in 250 μl of dimethylformamide. The mixturewas allowed to stand for 6 hours at ambient temperature, and was thenplaced in the refrigerator. After two days, the mixture was centrifuged,and the supernatant was chromatographed on Bio-Gel P6, eluting withphysiological buffered saline, to obtain 8.5 ml of solution, which wasanalyzed by ultraviolet, observing the spectrum at 280 and 370 nm. Theanalysis showed that the product had a conjugation ratio of 6.4 moles ofdrug per mole of antibody, and that the solution contained 0.64 mg/ml ofconjugate.

The product was evaluated for its ability to inhibit the growth of T222tumor cells in tissue culture. It was found that the conjugate produced70% inhibition of cell growth at 0.035 mcg/ml concentration, based onmethotrexate content.

Preparation 6

Antibody L/1C2 F(ab')₂ fragment

The F(ab')₂ fragment of antibody L/1C2 was prepared by adding 2.4 ml ofpepsin solution, containing 12.6 mg of pepsin/ml, to 1.5 g of L/1C2antibody in 270 ml of physiological buffered saline. The mixture washeld at 37° C. for 2 hours and 20 minutes, and then the reaction wasstopped by the addition of triethanolamine. The product was thenconcentrated by chromatography on a Sepharose Fast Flow column, elutingwith 0.15M sodium acetate. The F(ab')₂ -containing fractions werecombined, and concentrated by dialysis to obtain 100 ml of productsolution containing 992 mg of the F(ab')₂ fragment of antibody L/1C2.

Preparation 7

Antibody L/1C2 F(ab')₂ fragment propionyl-3-aminocarbonyl-4-benzaldehyde

L/1C2 F(ab')₂ fragment, prepared in Preparation 6, was dialyzed into0.34M borate buffer at pH 8.6 to obtain 23 mg (0.23 μmoles) of F(ab')₂fragment in the form of 3.8 ml of solution. That solution was combinedwith 0.44 mg (1.4 μmoles) of 3-(4-formylphenylcarbonylamino)propionicacid, N-succinimido ester, in 102 μl of acetonitrile. The mixture wasstirred for one hour at ambient temperature, and the solution was thenchromatographed over a column of 11 g of Sephadex G25, eluting with 0.1Msodium acetate at pH 5.6. The product-containing fractions were combinedto obtain 19 mg (0.19 μmoles) of derivatized antibody fragment, at aconjugation ratio of 2.8 moles per mole, in 9.6 ml of solution.

EXAMPLE 4 Conjugate of L/1C2 F(ab')₂ fragmentpropionyl-3-aminocarbonyl-4-benzaldehyde with methotrexate-γ-hydrazide

To 2.7 ml of the derivatized antibody fragment solution from Preparation7, containing 8 mg (0.08 μmoles) of derivatized antibody fragment, wasadded 0.47 ml of 1M phosphate buffer at pH 5.6. To that solution wasadded 3.7 mg (7.g μmoles) of methotrexate-γ-hydrazide, dissolved in theminimal amount of 0.1M sodium acetate. The pH of the mixture wasadjusted back to 5.6 with dilute hydrochloric acid, and the mixture wasstirred for 17 hours at ambient temperature. It was then centrifuged,and the supernatant was chromatographed on a column of 11 g of SephadexG25, eluting with physiological buffered saline. Ultraviolet analysisshowed that 5.8 mg of conjugate was obtained, in the form of a solutioncontaining 0.95 mg/ml, at a conjugation ratio of 2.1 moles per mole.

The conjugate was tested in tissue culture against T222 tumor cells, andwas found to inhibit growth of the cells to the extent of 22% at aconcentration of 0.0046 mcg/ml, and to inhibit growth by 83% at aconcentration of 0.046 mcg/ml, based on content ofmethotrexate-γ-hydrazide.

Preparation 8

3-(5-Formylpyrrol-2-ylcarbonylamino)propionic acid, N-succinimido ester

To a flask were added 139 mg of 5-formylpyrrol-2-ylcarboxylic acid, 247mg of dicyclohexylcarbodiimide, 138 mg of N-hydroxysuccinimide and 10 mlof dimethylformamide. The mixture was stirred for two hours at ambienttemperature under nitrogen, and the solvent was removed under vacuum toobtain 394 mg of crude active ester.

The above residue was taken up in 1 ml of acetonitrile. Not all of theresidue went into solution. The heterogeneous mixture was added slowlyto a solution of 89 mg of β-alanine in 2 ml of 0.5N sodium hydroxide.Concurrently, 1N sodium hydroxide was added to maintain the pH between7.5 and 8.0. The mixture was then stirred at ambient temperature for 1hour, and was filtered. The filtrate was made acid with dilutehydrochloric acid, was saturated with sodium chloride, and was extractedwith ethyl acetate. It was then dried and concentrated to an orange oil,which was chromatographed on a silica gel column, eluting with 10%methanol in dichloromethane. The product-containing fractions wereconcentrated under vacuum to obtain 49 mg of3-(5-formylpyrrol-2-ylcarbonylamino)propionic acid.

A 31 mg portion of the above intermediate was taken up in 3 ml ofdioxane, and to it was added 35 mg of dicyclohexylcarbodiimide and 19 mgof N-hydroxysuccinimide. The mixture was stirred for 2 hours at ambienttemperature under nitrogen, and was then filtered and concentrated undervacuum. The residue was chromatographed on silica gel, eluting with 5%methanol in dichloromethane to obtain about 6 mg of the desiredintermediate active ester.

Preparation 9

Antibody 007B propionyl-3-aminocarbonyl-2-pyrrol-5-carboxaldehyde

Antibody 007B is produced by a hybridoma which is a subclone derivedfrom the hybridoma producing the antibody KS1/4, which was described byVarki et al., Cancer Research 44, 681-86 (1984). A 105 mg portion ofantibody 007B, in the form of a solution containing 15 mg/ml in 0.34Mborate buffer, was combined with 300 μl of a solution of theintermediate of Preparation 8 containing 1.29 mg of the intermediateactive ester in acetonitrile. The mixture was stirred for 1 hour atambient temperature, and was then chromatographed over a 10 g column ofSephadex G25, eluting with 0.1M sodium acetate at pH 5.6. Theproduct-containing fractions were combined and analyzed by ultravioletspectroscopy, observing the curve at 280 and 300 nm. The productcomprised 14.6 ml of solution, at a concentration of 5.7 mg/ml, a totalof 83.2 mg of the intermediate named above. The conjugation ratio was3.6.

EXAMPLE 5 Conjugate of antibody 007B propionyl-3-aminocarbonyl-2-pyrrol-5-carboxaldehyde with methotrexate-γ-hydrazide

A 15 mg portion of the intermediate of Preparation 9, in the form of 3.1ml of a solution containing 4.8 mg/ml of a buffer at pH 5.6 containing0.1M sodium acetate and 0.15M phosphate ion, was combined with 4.7 mg ofmethotrexate-γ-hydrazide in 0.2 ml of sodium acetate buffer. The pH wasadjusted to 5.6 after the addition, and the mixture was stirred atambient temperature for about 24 hours. It was then centrifuged, and thesupernatant was chromatographed on a 10 g column of Sephadex G25,eluting with physiological buffered saline. The product-containingfractions were combined and filtered through an 0.22 micron filter toobtain 6.05 ml of product solution containing 9.9 mg of the desiredconjugate, at a conjugation ratio of 2.3, as determined by ultravioletspectroscopy, observing the curve at 250 and 280 nm. The bindingcapacity of the conjugate was assessed by radioimmunoassay, comparing itwith unconjugated 007B antibody. The titration curves of the conjugateand the antibody were substantially similar, indicating that the bindingcapacity of the antibody was essentially unchanged by conjugation withthe linker and drug.

Preparation 10

Antibody KS1/4 propionyl-3-aminocarbonylbutanal, diethylacetal

Antibody KS1/4 was dialyzed into 0.34M borate buffer at pH 8.6, at aconcentration of 20 mg/ml. One ml portions of the antibody solution werereacted with 3-(4,4-diethoxybutylaminocarbonyl)propionic acid,N-succinimido ester, which was added as a 20 mg/ml solution indimethylformamide. In one case the amount of active ester was 0.24 mg,and in the other case the amount was 0.48 mg. Both reaction mixtureswere stirred for 2 hours, and were then chromatographed over Biogel P6,eluting with physiological buffered saline.

The product of the first reaction was 17.8 mg of the desiredintermediate, and the product of the second reaction was 15.6 mg of thedesired intermediate.

EXAMPLE 6 Conjugate of antibody KS1/4 propionyl-3-aminocarbonylbutanal,diethylacetal with methotrexate-γ-hydrazide

One ml portions of the derivatized antibody made in Preparation 10 abovewere reacted with methotrexate-γ-hydrazide in 0.34M borate buffer at pH8.6. In the case of the first product of Preparation 10, 3.5 mg of theproduct above was reacted with 2.3 mg of methotrexate-γ-hydrazide as adimethylformamide solution. In the case of the second product, theamount of it was 2.9 mg and the amount of methotrexate-γ-hydrazide was2.0 mg. In each case the reaction mixture was then adjusted to pH 4.9.The reactions were then carried out for 18 hours at 37° , and theproducts were chromatographed on Biogel P6, eluting with physiologicalbuffered saline.

The product-containing fractions of each reaction were combined, andwere analyzed by ultraviolet spectroscopy, observing the curves at 279and 370 nm. The product of the first reaction was 2.7 mg of the desiredconjugate, at a conjugation ratio of 2.0. The product of the secondreaction was 2.6 mg, at a conjugation ratio of 3.9.

The conjugates of the present invention are useful in the method ofinhibiting the growth of unwanted cells which is an important part ofthe present invention. Accordingly, the invention also includes apharmaceutical composition, most preferably a parenteral compositionsuitable for injection into the body of the patient. Such compositionsare formulated by methods which are commonly used in pharmaceuticalchemistry. The present conjugates are acceptably soluble inphysiologically-acceptable fluids, such as physiological salinesolutions and other aqueous solutions which can safely be administeredparenterally.

Products for parenteral administration are often formulated anddistributed in solid, preferably freeze-dried form, for reconstitutionimmediately before use. Such formulations are useful compositions of thepresent invention. Their preparation is well understood bypharmaceutical chemists; in general, they comprise mixtures of inorganicsalts, to confer isotonicity, and dispersing agents, such as lactose, toallow the dried preparation to dissolve quickly upon reconstitution.Such formulations are reconstituted with highly purified water to aknown concentration, based on the methotrexate drug.

The optimum dosage and administration schedule of conjugates of thepresent invention must be determined by the treating physician, in thelight of the patient's condition.

It is customary, of course, to administer cytotoxic drugs in the form ofdivided doses, with intervals of days or weeks between each series ofdoses. The present conjugates are effective over a wide dosage range,and dosages per week will usually fall within the range from about 50 toabout 1000 mg/m² of methotrexate drug, more preferably in the range fromabout 200 to about 400 mg/m².

We claim:
 1. A derivatized antibody of the formula

    Ab[CO--X═R.sup.8 ].sub.m

wherein m is an integer from 1 to about 10; R⁸ is O, (OCH₃)₂ or (OCH₂CH₃)₂ ; Ab is a physiologically-acceptable antibody orantigen-recognizing fragment thereof, which recognizes an antigenassociated with an undesirable cell; X is a linker of the formula##STR7## R is hydrogen, phenyl, or phenyl substituted with one or twonitro, halogen, cyano or trifluoromethyl groups; Ar is ##STR8## R¹ ishydrogen, amino, amino-C₁ -C₄ alkyl, hydroxy-C₁ -C₄ alkyl, orguanidino-C₁ -C₄ alkyl; R² is (CH₂)_(n') ##STR9## n is an integer from 0to 5 provided when X is --(CH₂)_(n) --Ar--C═ and Ar is ##STR10## n maynot be
 0. 2. A derivatized antibody of claim 1 wherein the antibody is amonoclonal or chimeric antibody, or an antigen-recognizing fragmentthereof.
 3. A derivatized antibody of claim 2 wherein X is

    --(CH.sub.2).sub.n --Ar--CH═.


4. A derivatized antibody of claim 3 wherein m is from about 3 to about8.
 5. A derivatized antibody of claim 4 wherein the antibody is L/1C2.